Hemagglutinin proteins expressed on the surface of many viruses, such as influenza, rubella, smallpox, and others, agglutinate red blood cells (erythrocytes). This effect provides the basis for virus titration using hemagglutination (HA) assays. Specific attachment of antibodies to epitopes of the hemagglutinins responsible for attachment to the erythrocytes blocks binding of the virus particles to erythrocytes. This effect provides the basis for hemagglutination inhibition (HAI or HI) assays.
Hemagglutination assays and hemagglutination inhibition assays were introduced into medical and virology practice more than 60 years ago (Salk (1944) J. Immunol. 49, 87-98). Since that time, they have become important tools for measuring concentrations and strengths of viral cultures, the efficacy of the anti-viral immunization, and for studying the neutralizing capacity of virus-specific antibodies.
Two decades later, attempts were made to develop the method to a universal standard (Hierholzer et al. (1969) Applied Microbiol. 18, 824-833). However, the protocol for HAI assays kept undergoing minor modifications (e.g., Cross (2002) Seminars in Avian and Exotic Pet Medicine 11, 15-18; Hubby et al. (2007) Vaccine 25, 8180-8189; Wang et al. (2008) Vaccine 26 3626-3633; Noah et al. (2009), Clinical and Vaccine Immunology 16, 558-566), while preserving the core elements intact: observation of the agglutination in the solution volume, and visual detection of hemagglutination or hemagglutination inhibition.
In classical HA/HAI assays, the antigen (e.g., live or inactivated virus), either as is, or pre-incubated with an anti-serum or antibody of interest, is mixed with a suspension of purified erythrocytes, such as human group O erythrocytes, or avian, equine, or murine erythrocytes, depending on the type of the virus and objective of the study. After incubation of the mixture in V- or U-bottomed microwells, the major visual effect can be two-fold:                If antiserum is absent or unable to effectively block the attachment of the virus to erythrocytes, the virus particles link the erythrocytes into a dispersed three-dimensional semi-transparent gel, referred to as a “halo.”        If the virus is effectively blocked or absent, then the erythrocytes precipitate to the bottom of the well, forming the characteristic bright pellet, or “button.”        
Using avian erythrocytes, the agglutination effect can be observed (optionally) by inability of the agglutinated erythrocytes to flow down the V-surface of the tilted plates.
To determine the concentration or strength of a viral culture in the HA hemagglutination assay, the sample is subjected to two-fold serial dilutions, until the agglutination vanishes. To determine the efficacy of the antiserum or tested antibody in the HAI assay, the serum sample is similarly subjected to serial dilution, until agglutination appears. The last dilution on the “borderline” between agglutination/non-agglutination is called the HA or HAI titer.
HA and HAI assays are used for the study of immune response to a multitude of different pathogenic viruses, including adenoviruses, enteroviruses, reoviruses, myxoviruses, poxviruses, and flaviviruses, which cause a wide spectrum of human and animal illnesses, from influenza and rubella to smallpox and Dengue hemorrhagic fever (e.g., Hatgi et al. (1966) Am. J. Trop. Med. Hyg. 15, 601-610; Hierholzer et al. (1969) Applied Microbiol. 18, 824-833; Cross (2002) Seminars in Avian and Exotic Pet Medicine 11, 15-18; Hubby et al. (2007) Vaccine 25, 8180-8189; Wang et al. (2008) Vaccine 26, 3626-3633). Thus, HA and HAI tests remain major tools in modern virology (WHO Manual on Animal Influenza Diagnosis and Surveillance, WHO/CDS/CSR/NCS2002.5 Rev. 1.). Significant improvements to the assays could be of widespread benefit.
The virtues of HA/HAI assays, based on erythrocytes, inspired the development of various versions of agglutination/agglutination inhibition tests, based on latex microbeads coated with various antigens and affinity ligands, including hemagglutinins and virus particles (Ko et al. (1999), J. Clin. Pathol. 52, 770-772; Xu et al. (2005), J. Clin. Microbiol., 43, 1953-1955). These methods, although fast and reliable, do not provide greater sensitivity to sera or antibody solutions than the classical HAI.
An objective of the invention presented here was the development of a functional assay of enhanced sensitivity that would use real target cells (e.g., erythrocytes), rather than synthetic particles, and would stay as close as possible to the well-proven and widely accepted classical HA/HAI. On the other hand, basic principles illustrated in embodiments of the current invention could be applied to latex bead agglutination methods to increase their sensitivity and informational capacity.
While robust, uncomplicated and reliable, HA and HAI assays lack adequate sensitivity in the cases of some conditions, such as measles, yellow fever, and polyoma (Chapagain et al. (2006) Virology J, 3, 3-5; Fujino et al. (2007) J. Virological Methods 142, 15-20; Niedrig et al. (1999) Trop. Med. Int. Health 4, 67-71). Further, assessments of agglutination are typically performed by the human eye, which can become a source of subjective evaluation.
In addition to the inadequate sensitivity of the standard HA/HAI assays with many viruses, as mentioned above, the development of modern in vitro systems for high-throughput analysis of immune responses, such as the MIMIC® system, described in US 2005/0282148, required improved sensitivity in methods of evaluating functionality of antibody immune responses. The MIMIC® system is based on cultures of human immune-competent cells developed in a 96-well format, which limits the achievable concentrations and total quantities of the antigen-specific antibodies generated in the system.
Thus, there is a continuing need for functional assays with improved sensitivity, including those based on hemagglutination.